The cardiac mitochondrial K ATP channel (mitoK ATP) plays an essential role in cardioprotection against ischemia-reperfusion injury. Recent findings show that it also participates in the ouabain signaling pathway. The central hypothesis of this project is that ouabain-activated cytosolic signals are relayed to mitoKATP, resulting in increased production of reactive oxygen species (ROS) and cardiac protection. Our long-term goal is to understand the biological significance of mitoKATP in regulation of cell growth, gene expression and cellular function. Aim 1 will test the underlying hypothesis by demonstrating that ouabain-induced ROS production requires opening of mitoK ATP and determining the mechanism by which mitoK ATP opening causes increased ROS production. Aim 2 will examine whether RACKs form a signaling platform that transmits the ouabain signal to PKC isozymes and Raf. Aim 3 will study translocation of signaling proteins to mitochondria and subsequent increases in mitochondrial protein phosphorylation. Aim 4 will test the hypothesis that mitoK ATP opening forms a positive signal amplification loop for ouabain-activated signal transduction. Aim 5 is designed to reveal the physiological significance of the interactions between ouabain and mitoK ATP in ischemic cardioprotection and inotropy. The experimental approaches include measurements of K+ flux through purified mitoK ATp, bioenergetic studies on mitochondria, physiological studies on cardiomyocytes and perfused hearts, and biochemical studies on the signaling pathways of cardiomyocytes.